Protein melt analysis using dipyrrometheneboron difluoride compounds

ABSTRACT

According to the present teachings, systems, compositions, kits and methods for protein melt analysis are provided that utilizing a dye that is a dipyrrometheneboron difluoride compound. In some embodiments, a method comprises preparing a sample by mixing at least one protein with a dye, and applying a controlled heating, while recording the fluorescence emission of the sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/516,545 filed Jul. 19, 2019, which is a continuation of U.S.application Ser. No. 15/392,295 filed Dec. 28, 2016, which is adivisional of U.S. application Ser. No. 13/751,030 filed Jan. 25, 2013,now, U.S. Pat. No. 9,568,478, which claims a priority benefit under 35U.S.C. § 119(e) to U.S. Provisional Application No. 61/591,383 filedJan. 27, 2012, the contents of which are incorporated herein byreference in their entirety.

BACKGROUND

Proteins are typically the key molecule studied as the drug target fordrug development generation. High throughput screening of small-moleculeand ligand libraries that bind to protein targets is an important partof the process—requiring screening of thousands of small molecules andligands with a variety of different assays, requiring months of time.Protein targets are challenging to work with due to their susceptibilityto degradation and aggregation, so protein stability screening is oftenan important component of lead generation programs. Protein stabilityscreening, performed using the protein melting method, is employed inother research programs that involve native proteins. Protein melting isan extremely useful screening method for the identification of ligandsand/or solution (buffer) conditions that maximally stabilize a proteinas part of protein purification, crystallization, and functionalcharacterization.

Historically, the methodologies to perform protein melt screening areeither very slow and tedious, analyzing one sample at a time—or ifhigh-throughput, require milligram amounts of protein sample and incurhigh costs in either reagents, or protein samples, or both. It would beuseful to have new and useful systems, methods and reagents to screenproteins, including antibodies, to identify ligands,mutations/modifications, buffer conditions, or other factors that affecttheir melting temperature (Tm) and relative stability.

SUMMARY OF THE DISCLOSURE

Various embodiments of systems and methods for protein melt analysisaccording to the present teachings provide for the determination ofprotein melt temperature (T_(m)) using species of a dye given by theparent structure:

-   -   4,4-difluoro-8-(maleimidylphenyl)-4-bora-3a,4a-diaza-s-indacene

wherein:

-   -   R1 to R6 may independently be —H, halogen, —(CH₂)_(n)CO₂H        (wherein n=0 to 6), —(CH₂)_(n)CO₂R (wherein n=0 to 6),        cycloakyl, alkyl (1-5 carbons), aryl, heteroaryl, arylalkyl        (wherein the alkyl portion is 1-5 carbon atoms), alkenyl, azido,        alkynyl, and sulfo; alone or in combination. Substituents o, m        and p are independently selected from hydrogen, halogen,        —(CH₂)_(n)CO₂H (wherein n=0 to 6), —(CH₂)_(n)CO₂R (wherein n=0        to 6), cycloakyl, alkyl (1-5 carbons), aryl, heteroaryl,        arylalkyl (wherein the alkyl portion is 1-5 carbon atoms),        alkenyl, azido, alkynyl, sulfo, and a maleimidyl substituent        having the structure:

-   -   alone or in combination with the proviso that the maleimidyl        substituent occurs in formula (I) once and only once. An alkenyl        substituent may be substituted or unsubstituted, wherein the        alkenyl group is ethenyl, dienyl, or trienyl. Substituents for        an alkenyl group may be selected from hydrogen, halogen, alkyl        (1-5 carbon atoms), cyano, carboxylate ester, carboxamide, aryl,        or heteroaryl. Aryl may be selected from phenyl, 1-naphthyl,        2-naphthyl, 1-pyrenyl, 9-anthryl, pyridyl, quinolyl, and alkoxy        derivatives thereof. Any aryl group in any substituent may be        further substituted by halogen, (CH₂)_(n)CO₂H (wherein n=0 to        6), —(CH₂)_(n)CO₂R (wherein n=0 to 6) alkyl (1-5 carbons), and        alkoxy(wherein the alkyl portion is 1-4 carbon atoms). Any alkyl        group in any substituent of an aryl group may be further        substituted by an ester or amide substituent. L is an optionally        present linker. A linker may be selected from alkyl (1-6        carbons), and heteroalkyl, (1-6 atoms).

Non-limiting examples of a species according to various embodiments ofsystems and methods may be selected from:

4,4-difluoro-1,3,5,7-tetramethyl-8-(4-maleimidylphenyl)-4-bora-3a,4a-diaza-s-indacene(BODIPY® 499/508 maleimide), and from:

4,4-difluoro-3,5-bis(4-methoxyphenyl)-8-(4-maleimidylphenyl)-4-bora-3a,4a-diaza-s-indacene(BODIPY® 577/618 maleimide).

Various embodiments of systems and methods of the present teachingsutilize various embodiments of a dye of formula (1) to monitor proteinfolding. In various systems and methods of the present teachings, amaleimide substituted phenyl ring in position 8 of thedipyrrometheneboron difluoride ring may react selectively with a thiolgroup of, for example, but not limited by, a naturally occurringcysteine residue in a protein to form a covalent C—S bond between adipyrrometheneboron difluoride dye species as depicted in formula (1)and the cysteine residue. A protein so selectively modified may then bethermally denatured, so that the dye tag may be used to monitor theprogress of the thermal denaturation, and a melting temperature (T_(m))may then be determined from the data so generated.

According to various embodiments, the stability of the dye and of thedye-thiol bond may provide for a wide variety of assay conditions underwhich a protein melt analysis may be run, thereby providing for a widenumber of types of proteins and types of thermal melt assays that may beperformed. For various embodiments of the present teachings, a speciesof a dye of formula (1) may be selected so that it has an excitationwavelength of between about 470 nm to about 650 nm, and an emissionwavelength between about 500 nm to about 700 nm. As one of ordinaryskill in the art is apprised, proteins may be damaged by irradiation inthe UV. Therefore, for the purpose of protein folding study, a dyeselected having such excitation/emission characteristics may beadvantageous for preserving a protein structure, and therefore providinga consistent protein melt determination. Various embodiments of a dyeselected from formula (1) may exhibit enhanced stability towardsphotobleaching, providing for stable signals solely related to theprotein folding process over an entire protein melt analysis. Forvarious embodiments of systems and methods of the present teachings, thethiol group may be an intrinsic feature of a cysteine-containingprotein. In various embodiments of systems and methods of the presentteachings, a protein or proteins may be specifically tagged with atthiol group to provide for reactivity with a dye selected from formula(1). According to various embodiments, the selectivity of the binding ofthe dye to cysteine residues in a protein may be useful fordiscriminating the progress of reaction for cysteine-containing proteinsin a mixture of proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram of a PCR instrument according to variousembodiments of methods of the present teachings.

FIG. 2 is a block diagram that illustrates components of an exemplarycomputer system that may be utilized in the control and interface of thePCR instrumentation according to various embodiments of methods of thepresent teachings.

FIG. 3A is a graph of protein thermal shift (PTS) data forβ-lactoglobulin (bovine) using a dye according to various embodiments ofsystems and methods of the present teachings in comparison to dye knownin the art of PTS.

FIG. 3B shows the first derivative graphs of the β-lactoglobulin PTSdata shown for FIG. 3A.

FIG. 4A is the graph of PTS data for β-lactoglobulin (bovine) shown inFIG. 3A, which is an expanded view of the details of the background forthe dye known in the art of PTS.

FIG. 4B depicts the first derivative graphs of the β-lactoglobulin PTSdata shown for FIG. 3A.

FIG. 5A is a graph of PTS data for α-chymotrypsin (bovine) using a dyeaccording to various embodiments of systems and methods of the presentteachings in comparison to dye known in the art of PTS.

FIG. 5B shows the first derivative graphs of the α-chymotrypsin PTS datashown for FIG. 5A.

FIG. 6A is the graph of PTS data for α-chymotrypsin (bovine) shown inFIG. 5A, which is an expanded view of the details of the background forthe dye known in the art of PTS.

FIG. 6B depicts the first derivative graphs of the α-chymotrypsin PTSdata shown for FIG. 5A.

FIG. 7A is a graph of PTS data for exonuclease I (E. coli) using a dyeaccording to various embodiments of systems and methods of the presentteachings in comparison to dye known in the art of PTS.

FIG. 7B shows the first derivative graphs of the exonuclease I PTS datashown for FIG. 7A.

FIG. 8A is the graph of PTS data for exonuclease I (E. coli) shown inFIG. 7A, which is an expanded view of the details of the background forthe dye known in the art of PTS.

FIG. 8B depicts the first derivative graphs of the exonuclease I PTSdata shown for FIG. 7A.

FIG. 9A is a graph of PTS data for lysozyme (ovine) using a dyeaccording to various embodiments of systems and methods of the presentteachings in comparison to dye known in the art of PTS.

FIG. 9B shows the first derivative graphs of the lysozyme PTS data shownfor FIG. 9A.

FIG. 10A is a graph of PTS data for a CAAX-containing membrane proteinusing a dye according to various embodiments of systems and methods ofthe present teachings in comparison to dye known in the art of PTS.

FIG. 10B shows the first derivative graphs of the CAAX-containingmembrane protein PTS data shown for FIG. 10A.

DETAILED DESCRIPTION

The present teachings relate to embodiments of systems and methodsproviding for a dye of formula (1) useful for protein melt curveanalysis:

-   -   4,4-difluoro-8-(maleimidylphenyl)-4-bora-3a,4a-diaza-s-indacene

wherein:

-   -   R1 to R6 may independently be —H, halogen, —(CH₂)_(n)CO₂H        (wherein n=0 to 6), —(CH₂)_(n)CO₂R (wherein n=0 to 6),        cycloakyl, alkyl (1-5 carbons), aryl, heteroaryl, arylalkyl        (wherein the alkyl portion is 1-5 carbon atoms), alkenyl, azido,        alkynyl, and sulfo; alone or in combination. Substituents o, m        and p are independently selected from hydrogen, halogen,        —(CH₂)_(n)CO₂H (wherein n=0 to 6), —(CH₂)_(n)CO₂R (wherein n=0        to 6), cycloakyl, alkyl (1-5 carbons), aryl, heteroaryl,        arylalkyl (wherein the alkyl portion is 1-5 carbon atoms),        alkenyl, azido, alkynyl, sulfo, and a maleimidyl substituent        having the structure:

alone or in combination, with the proviso that the maleimidylsubstituent occurs in formula (I) once and only once. An alkenylsubstituent may be substituted or unsubstituted, wherein the alkenylgroup is ethenyl, dienyl, or trienyl. Substituents for an alkenyl groupmay be selected from hydrogen, halogen, alkyl (1-5 carbon atoms), cyano,carboxylate ester, carboxamide, aryl, or heteroaryl. Aryl may beselected from phenyl, 1-naphthyl, 2-naphthyl, 1-pyrenyl, 9-anthryl,pyridyl, quinolyl, and alkoxy derivatives thereof. Any aryl group in anysubstituent may be further substituted by halogen, —(CH₂)_(n)CO₂H(wherein n=0 to 6), —(CH₂)_(n)CO₂R (wherein n=0 to 6) alkyl (1-5carbons), and alkoxy(wherein the alkyl portion is 1-4 carbon atoms). Anyalkyl group in any substituent of an aryl group may be furthersubstituted by an ester or amide substituent. L is a linker that isoptionally present. According to various embodiments, a linker may beselected from alkyl (1-6 carbons), and heteroalkyl, (1-6 atoms).

According to various embodiments, a melting temperature (T_(m)) may bedetermined from a protein denaturation study under wide range of assayconditions using various dipyrrometheneboron difluoride dyes accordingto formula (1). In various embodiments of systems and methods accordingto the present teachings, such a range of assay conditions may include,but not limited by, a wide pH range useful in protein folding studiesfrom about pH 2 to about pH 10, a wide variety of buffer selections andconcentrations, and wide variety of other assay constituents, such asvarious salts of the Hofmeister series, various surfactants, as well asvarious protein stabilizing agents, such as polysaccharides and otherpolyols of a wide range of molecular weights and concentrations.According to the present teachings, various dipyrrometheneborondifluoride dyes according to formula (1) have excitation wavelengths ofbetween about 470 nm to about 650 nm, and an emission wavelength betweenabout 500 nm to about 700 nm. Accordingly, using dyes selected fromformula (1) avoids irradiation of protein samples with UV, therebyavoiding artifacts due to protein photo-degradation. Various embodimentsof dipyrrometheneboron difluoride dyes according to formula (1) may alsoexhibit enhanced stability towards photobleaching, providing for stablesignals solely related to the protein folding process. Accordingly,dipyrrometheneboron difluoride dyes according to formula (1) haveattributes that make them useful for a wide variety of protein meltassays.

Non-limiting examples of a species according to various embodiments ofsystems and methods may be selected from:

4,4-difluoro-1,3,5,7-tetramethyl-8-(4-maleimidylphenyl)-4-bora-3a,4a-diaza-s-indacene(BODIPY® 499/508 maleimide), and from:

4,4-difluoro-3,5-bis(4-methoxyphenyl)-8-(4-maleimidylphenyl)-4-bora-3a,4a-diaza-s-indacene(BODIPY® 577/618 maleimide)

As used herein, “substituted” refers to a molecule wherein one or morehydrogen atoms are replaced with one or more non-hydrogen atoms,functional groups or moieties. For example an exemplary unsubstitutedethenyl group may be represented —CH═CH—. Substituted ethenyl groups mayinclude, for example, but are not limited by, —CH═CH—COOH, —CH═CHCOOR,—CH═CH-Aryl and —CH═CH-Aryl-OR where, for example, R is alkyl orsubstituted alkyl. As one of ordinary skill in the art is apprised, fora variety of organic dye molecules, some non-limiting exemplarysubstituents include, hydrogen, halogen alkyl, cycloalkyl, branchedalkyl, alkene, cyclic alkene, branched alkene, alkyne, branched alkyne,carboxyl, ester, sulfate, sulfonate, sulfone, amino, ammonium, amido,nitrile, alkoxy, phenoxy, phenyl, polycyclic aromatic, and electron-richheterocycle. As will be discussed in more detail subsequently,non-limiting examples of substituents for various embodiments of dyes ofthe present teachings may include hydrogen, halogen, acid, ester,cycloakyl, alkyl, aryl, heteroaryl, arylalkyl, alkenyl, azido, alkynyl,and sulfo; alone or in combination. Such substituents by themselves besubstituted by non-limiting examples such as hydrogen, halogen, alkyl,cyano, carboxylate ester, carboxamide, aryl, or heteroaryl.

One of ordinary skill in the art may recognize various assays utilizingthe determination of the melting temperature (T_(m)) of a protein. Theprocess in which a protein having, for example, a tertiary structuregoes from a tertiary structure to a random coil structure is referred toin the art as, for example, but not limited by, protein denaturation,protein unfolding, and protein melt. Additionally, a protein undervarious sample solution conditions may show a variation or shift in theobserved T_(m) for that protein as a function of the sample solutionconditions. Various terms such as thermal melt assays (TMA), thermalshift assay (TSA), protein thermal shift (PTS) analysis, anddifferential scanning fluorimetry (DSF) are examples of terms of the artin which the determination of the T_(m) of a protein or proteins iscentral to the analysis.

In addition to the determination of a melting temperature (T_(m)),various embodiments of isothermal denaturation (ITD) may be utilized, inwhich a time to denaturation (D_(t)) is determined. For example, in someembodiments of ITD, a thermal ramp may be applied to a protein sampleunder a set of baseline sample solution conditions (pH, salt, ligand,etc.), and the Tm determined for those conditions. In a subsequentexperiment or set of experiments, a protein sample may be subjected tovarious sample solution conditions. However, for the subsequentanalysis, instead of a temperature ramp, the temperature determined forthe baseline sample solution conditions would be used in the experiment,and the fluorescence signal would be monitored as a function of time.The experiment may be repeated at temperatures close to thepredetermined T_(m) in order to compare the rate of denaturation as afunction of time and sample solution conditions.

With respect to aspects of measurement science applied to proteinchemistry, a change in detector signal amplitude may be observed as afunction of the change in the folded state of a protein. In that regard,various analyses may be based on either the increase or decrease offluorescence signal amplitude as it varies with respect to a temperatureor change in temperature applied to a protein sample.

For example, in various analyses, the signal amplitude may arise from anamino acid residue of the protein, such as tryptophan. As one ofordinary skill in the art is apprised, the intensity, quantum yield, andwavelength of maximum fluorescence emission of tryptophan are verysolvent dependent. The fluorescence spectrum shifts to shorterwavelength and the intensity of the fluorescence increases as thepolarity of the solvent surrounding the tryptophan residue decreases.Therefore, as a protein unfolds, buried tryptophan residues may beexposed to a more polar aqueous solvent environment, so that adecreasing signal amplitude may be observed from a folded to an unfoldedstate.

Instead of using an intrinsic signal arising from a protein molecule,other analyses may utilize a dye to indicate a folded state of aprotein. For example, a fluorescence dye, such as Sypro® Orange, may beutilized to monitor the folded state of a protein. For Sypro® Orange ina polar solvent environment, quenching of the fluorescent signal isobserved. For Sypro® Orange associated with the surface groups of afolded protein in solution, the dye is in an aqueous environment, sothat its fluorescence signal is quenched. As a protein is unfolded,using for example, thermal unfolding, hydrophobic regions or residuesmay be exposed. Sypro® Orange may then bind to hydrophobic regions orresidues, and fluorescence may thereby be increased. For such a Sypro®Orange assay, then an increasing signal amplitude going from a folded tounfolded state may be observed. Dyes, such as1-anilinonaphthalene-8-sulfonic acid (1,8-ANS) and4,4′-Dianilino-1,1′-Binaphthyl-5,5′-Disulfonic Acid (Bis-ANS), which arequenched in aqueous environments, have been shown to be useful formonitoring protein folding, in which the fluorescence of 1,8 ANS andBis-ANS may increase substantially in the process of, for example,protein refolding.

Monitoring protein thermal stability may be done in both academe, aswell as industry for a variety reasons. For example, but not limited by,protein melt curve studies, or thermal studies, may be done forinvestigation of mutations to a target protein as a result of, forexample, site directed mutagenesis studies. Additionally, proteinthermal stability studies may be done to screen for the impact onprotein stability due to a variety in vitro processing and storageconditions. Such protein thermal stability studies may screen for theimpact that a variety of additives, such as, buffers, ligands, andorganic agents may have on the thermal stability of the protein ofinterest. High throughput screening of the binding of drug candidates toprotein targets may also be monitored by the impact that the binding ofa drug candidate may have on protein thermal stability. Accordingly,identifying the conditions that affect protein thermal stability mayenhance the identification of a variety of desired conditions impactingprotein purification, crystallization, and functional characterization.

As will be discussed in more detail subsequently, various embodiments ofsystems and methods may utilize detector signal data collected over theentirety of a defined temperature range for a protein melt assay.According to various embodiments of methods and compositions of thepresent teachings, a dye according to formula (1) may be used in variousanalyses in which the determination of a protein T_(m) is desired. Insuch analysis, an apparatus capable of applying a controlled thermalramp and well-controlled isothermal heating, as well as detecting thesignal from a plurality of samples may be used in the determination ofprotein thermal melt analysis.

According to various embodiments of a thermal cycler instrument 100, asshown in FIG. 1, may be useful providing thermal control and detectionfor protein thermal melt analysis. A thermal cycling instrument, asdepicted in FIG. 1, may include a heated cover 114 that is placed over aplurality of samples 116 contained in a sample support device. Someexamples of a sample support device may include, but are not limited to,tubes, vials, and a multi-well plate permitting a selection of samplecapacities, such as a standard microtiter 96-well, and 384-well plate.In various embodiments, a sample support device may be a micro devicecapable of processing thousands of samples per analysis, such as variousmicrofluidic devices, microcard devices, and micro chip devices. Invarious embodiments, a sample support device may be a fabricated from asubstantially planar support, such as a glass, metal or plastic slide,having a plurality of sample regions. The sample regions in variousembodiments of a sample support device may include through-holes,depressions, indentations, and ridges, and combinations thereof,patterned in regular or irregular arrays formed on the surface of thesubstrate. In various embodiments, a sample support device may have acover between the sample regions and heated cover 114. A sample supportdevice may have sample regions arranged in a sample array format. One ofordinary skill in the art will recognize that many examples of a samplesupport device are patterned in row and column arrays. A sample arrayformat according to the present teachings may include any pattern ofconvenient and addressable arrangement of sample regions in a samplesupport device, including a single row or column of sample regions in asample support device.

In various embodiments of a thermal cycler instrument 100, include asample block 118, an element or elements for heating and cooling 120,and a heat exchanger 122. Various embodiments of a thermal blockassembly according to the present teachings comprise components 118-122of thermal cycler system 100 of FIG. 1.

In FIG. 1, various embodiments of a thermal cycling system 100 have thecomponents of embodiments of thermal cycling instrument 100, andadditionally a detection system comprising, an imager 110 and optics112. It should be noted that while a thermal cycler system 100 isconfigured to detect signals from samples 116 in a sample support deviceduring an analysis, a detection system according to the presentteachings may be used to detect signals from a sample support deviceafter an analysis has been completed.

A detection system may have an electromagnetic radiation source thatemits electromagnetic energy, and a detector or imager 110, forreceiving electromagnetic energy from samples 116 in sample supportdevice. A detector or imager 110 may capable of detectingelectromagnetic energy from samples 116 may a charged coupled device(CCD), back-side thin-cooled CCD, front-side illuminated CCD, a CCDarray, a photodiode, a photodiode array, a photo-multiplier tube (PMT),a PMT array, complimentary metal-oxide semiconductor (CMOS) sensors,CMOS arrays, a charge-injection device (CID), CID arrays, etc. Thedetector can be adapted to relay information to a data collection devicefor storage, correlation, and manipulation of data, for example, acomputer, or other signal processing system. Additionally, optics 112 ofa detection system may include components, such as, but not limited by,various positive and negative lenses, mirrors, and excitation andemission filters.

Regarding various embodiments of an electromagnetic radiation source fora detection system, such sources may include but are not limited to,white light, halogen lamps, lasers, solid state lasers, laser diodes,micro-wire lasers, diode solid state lasers (DSSL), vertical-cavitysurface-emitting lasers (VCSEL), LEDs, phosphor coated LEDs, organicLEDs (OLED), thin-film electroluminescent devices (TFELD),phosphorescent OLEDs (PHOLED), inorganic-organic LEDs, LEDs usingquantum dot technology, LED arrays, an ensemble of LEDs, floodlightsystems using LEDs, and white LEDs, filament lamps, arc lamps, gaslamps, and fluorescent tubes. Light sources can have high radiance, suchas lasers, or low radiance, such as LEDs. The different types of LEDsmentioned above can have a medium to high radiance.

Multiple excitation and emission filter sets can be employed in existingthermal cycling devices, wherein each filter set may includepre-selected excitation and emission filters to provide an accurateresponse of signal proportional to oligonucleotide concentration in asample at various stages of PCR. The excitation filter in a coupled setof filters can be chosen to allow wavelengths of light received from thelight source that are close to the peak excitation wavelength of apredetermined dye to pass. The excitation filter can also be configuredto block wavelengths of light that are greater than and less than thepeak excitation wavelength. Similarly, the emission filter in the set offilters can be chosen to allow light close to the peak emissionwavelength to pass while also blocking wavelengths outside the peakemission wavelength. In such a fashion, and as will be discussed in moredetail subsequently, a selection of spectrally distinguishable dyespecies, in conjunction with the detection system, and data processingcapabilities of a thermal cycling apparatus may provide for detection ofa plurality of dye signals in, for example, a multiplex assay.

In use, a detection system for use with a thermal cycling device mayfunction by impinging an excitation beam from an electromagneticradiation source on samples in a sample support device, therebygenerating a fluorescent radiation from the plurality of samples 116.Light emitted from the samples 116, may be transmitted through a lens orlenses, such as a well lens, a Fresnel lens, or a field lens, and thenmay be directed to additional optical components, such as a dichroicmirror, and an emission filter. Undesired wavelengths of light emittedfrom samples 116, may be reflected by the dichroic mirror or are blockedby the emission filter. A portion of the emitted light that transmitsthrough the dichroic mirror and emission filter may be received by adetector or imager 110. As previously mentioned, for a thermal cyclersystem 100, a detector or imager may generate data signals from thefluorescent radiation from the samples over time, or may generate datasignals from the fluorescent radiation from the samples at thecompletion of various analyses or assays. For various embodiments ofsystems and methods according to the present teachings, protein meltcurve data is acquired over time, and a T_(m) may be determined for eachsample.

Accordingly, though a thermal cycler instrument may be a useful platformfor the generation and acquisition of protein melt curve data, one ofordinary skill in the art would recognize that an instrument havingdetection and sample thermostatting capabilities may be useful forgenerating protein melt curve data.

For embodiments of thermal cycler instrumentation 100, a control system124, may be used to control the functions of the detection, heatedcover, and thermal block assembly. The control system may be accessibleto an end user through user interface 124 of thermal cycler instrument100. A computer system 200, as depicted in FIG. 2 may serve as toprovide the control the function of a thermal cycler instrument, as wellas the user interface function. Additionally, computer system 200 mayprovide data processing, as well as, with other components, provide fordisplay, and report preparation functions. For example, signals receivedby a detector or imager may be processed by various algorithms, such asa spectral deconvolution algorithm, which may then be displayed to anend user, as well as providing a report. All such instrument controlfunctions may be dedicated locally to the thermal cycler instrument, orcomputer system 200 may provide remote control of part or all of thecontrol, analysis, and reporting functions, as will be discussed in moredetail subsequently.

FIG. 2 is a block diagram that illustrates a computer system 300,according to various embodiments, upon which embodiments of thermalcycler system 100 of FIG. 1 may utilize. Computer system 200 includes abus 202 or other communication mechanism for communicating information,and a processor 204 coupled with bus 202 for processing information.Computer system 200 also includes a memory 206, which can be a randomaccess memory (RAM) or other dynamic storage device, coupled to bus 202for instructions to be executed by processor 204. Memory 206 also may beused for storing temporary variables or other intermediate informationduring execution of instructions to be executed by processor 204.Computer system 200 further includes a read only memory (ROM) 208 orother static storage device coupled to bus 202 for storing staticinformation and instructions for processor 204. A storage device 210,such as a magnetic disk or optical disk, is provided and coupled to bus202 for storing information and instructions.

Computer system 200 may be coupled via bus 202 to a display 212, such asa cathode ray tube (CRT) or liquid crystal display (LCD), for displayinginformation to a computer user. An input device 214, includingalphanumeric and other keys, is coupled to bus 202 for communicatinginformation and command selections to processor 204. Another type ofuser input device is cursor control 216, such as a mouse, a trackball orcursor direction keys for communicating direction information andcommand selections to processor 204 and for controlling cursor movementon display 212. This input device typically has two degrees of freedomin two axes, a first axis (e.g., x) and a second axis (e.g., y), thatallows the device to specify positions in a plane. A computer system 200provides data processing and provides a level of confidence for suchdata. Consistent with certain implementations of the present teachings,data processing and confidence values are provided by computer system200 in response to processor 204 executing one or more sequences of oneor more instructions contained in memory 206. Such instructions may beread into memory 206 from another computer-readable medium, such asstorage device 210. Execution of the sequences of instructions containedin memory 206 causes processor 204 to perform the process statesdescribed herein. Alternatively hard-wired circuitry may be used inplace of or in combination with software instructions to implementvarious embodiments of methods and compositions of the presentteachings. Thus implementations of the present teachings are not limitedto any specific combination of hardware circuitry and software.

The term “computer-readable medium” as used herein refers to any mediathat participates in providing instructions to processor 204 forexecution. Such a medium may take many forms, including but not limitedto, non-volatile media, volatile media, and transmission media.Non-volatile media includes, for example, optical or magnetic disks,such as storage device 210. Volatile media includes dynamic memory, suchas memory 206. Transmission media includes coaxial cables, copper wire,and fiber optics, including the wires that comprise bus 202.Transmission media can also take the form of acoustic or light waves,such as those generated during radio-wave and infra-red datacommunications. Common forms of computer-readable media include, forexample, a floppy disk, a flexible disk, hard disk, magnetic tape, orany other magnetic medium, a CD-ROM, any other optical medium, punchcards, paper tape, any other physical medium with patterns of holes, aRAM, PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge,a carrier wave as described hereinafter, or any other medium from whicha computer can read.

Further, it should be appreciated that a computer 200 of FIG. 2 may beembodied in any of a number of forms, such as a rack-mounted computer, adesktop computer, a laptop computer, or a tablet computer. According tovarious embodiments of a computer 200 of FIG. 2, a computer may beembedded in any number of mobile and web-based devices not generallyregarded as a computer but with suitable processing capabilities.Example of such devices may include, but are not limited by a PersonalDigital Assistant (PDA), a smart phone, and a notepad or any othersuitable electronic device. Additionally, a computer system can includea conventional network system including a client/server environment andone or more database servers. A number of conventional network systems,including a local area network (LAN) or a wide area network (WAN), andincluding wireless and wired components, are known in the art.Additionally, client/server environments, database servers, and networksare well documented in the art.

As previously discussed, a dye selected from members of the family ofdyes represented by formula (1) above have a number of desirableattributes that make them useful candidates as a dye for protein meltanalysis. Various embodiments of a dye according to formula (1) have anexcitation wavelength of between about 470 nm to about 650 nm, and anemission wavelength between about 500 nm to about 700 nm. Though notmeaning to provide a complete description of the physical properties ofdipyrrometheneboron difluoride dyes of formula (1), the phenyl ringsubstituent at position 8 of the dipyrrometheneboron difluoride ringstructure from which the maleimide group is appended, provides formodulation of auto-fluorescence of dyes of formula (1). In that regard,it has been observed that the strongest modulation of auto-fluorescencefor dyes of formula (1) may occur with the maleimide substituent is inthe ortho or para position of the phenyl ring. For example, dye species(1a) and (1b) exhibit negligible background fluorescence. Moreover, withrespect to the linker, Lx, the absolute length of the linker may beselected based on the impact to the modulation of the maleimide linkedgroup to the phenyl ring. Generally, the longer the linker becomes, adecrease in modulation of the background fluorescence is expected. Inthat regard, a linker must be judiciously selected to optimizesufficiently low background fluorescence.

The stability of dipyrrometheneboron difluoride dyes of formula (1) to arange of assay conditions provides compatibility with a range of proteinmelt assays. The dye has intrinsic pH stability between about pH 2 toabout pH 10, which is well within a range of useful pH conditions forprotein melt studies. With respect to aqueous solubility, generally, astock solution of a dye may be prepared in a variety of polar solventssuch as, but not limited by, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), acetonitrile, methanol, ethanol, and isopropanol. Onceprepared in a stock solution in a polar solvent, the dyes may beprepared in a selected assay solution, which may be a buffered solutionwith any number of additives, for example, but not limited by, thoseenhancing protein stability.

As a general thumb rule, the only solution conditions that would be notfavorable for the use of the dye of formula (1) for a variety of proteinmelt analyses would be a reactive non-protein nucleophile additive thatwould react with a dye and not with the intended protein target. In thatregard, any of a wide variety of organic and inorganic buffers over anyof a desirable range of buffer concentrations may be used withdipyrrometheneboron difluoride dyes of formula (1). Additionally, a widevariety of additives, for example, but not limited by, proteinstabilizing additives, may be used with dipyrrometheneboron difluoridedyes of formula (1). For example, it is known that membrane proteins aregenerally stabilized in vitro in concentrations of surfactants at orabove the critical micelle concentration (CMC). Any variety ofsurfactants, such as, but not limited by, the alkyl saccharidesurfactants (e.g. dodecyl-β-D-maltoside (DDM); octyl-β-D-glucoside(ODG)), polysorbate surfactants (e.g. Tween 20;Tween 80), fluorinatedsurfactants (e.g. perfluoralkyl acids), polyoxyethylene surfactants(e.g. Brij 56;Brij 58), polyethoxylated phenol surfactants (e.g.NP-40;Triton X100), anionic surfactants (e.g. methyl ester sulfonate(MES); alcohol ether sulfate (AES)), cationic surfactants (e.g.(cetyltrimethylammonium bromide (CTAB)) and zwitterionic surfactants(e.g. CHAPS, CHAPSO, Big-CHAP), have been recognized as havingproperties useful for stabilizing various membrane proteins. Withrespect to other classes of proteins, for example, many proteinsisolated from a variety of biological sources, are known to bestabilized in vitro using salts of the Hofmeister series, such as sodiumchloride, potassium chloride, ammonium phosphate, and ammonium sulfate.Mono- and poly-saccharides, such as sucrose, maltose, trehalose,dextrose and sorbitol, as well as well as a number of other polyols,such as glycerol, are known to stabilize various proteins in vitro. Anyof the above referenced assay conditions may be used withdipyrrometheneboron difluoride dyes of formula (1) for protein meltassays.

In FIG. 3A and FIG. 3B depicts protein melt data and first derivative ofthe melt data, respectively, for a sample of bovine β-lactoglobulin(Sigma PN L8005). The protein was prepared in phosphate buffered saline(PBS; 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, and a pH of 7.4) and 5%glycerol at a concentration of 3 mg/ml of protein. A 500× stock of a dyeof formula (1a); BODIPY® 499/508 maleimide, was prepared at 4 mg/ml inDMSO. The dye stock solution was diluted using a protein thermal shift(PTS) phosphate buffer (250 mM, pH 7.0) to a 10× stock solution. Theprotein-dye reaction mixture was prepared by using 1 μl of the proteinsolution, 2 μl of the 10× dye solution, and 17 μl of the PTS buffer. Tocompare to the results obtained using BODIPY® 499/508 maleimide,β-lactoglobulin samples were prepared using Sypro® Orange. The Sypro®Orange was prepared as a 1000× stock from a 5000× stock solution inDMSO, then diluted to a 10× stock solution using the PTS buffer. TheSypro® Orange-protein reaction mixture was prepared by using 1 μl of theprotein solution, 2 μl of the 10× dye solution, and 17 μl of the PTSbuffer. Triplicate samples for each protein-dye mixture were run on aViiA™ 7 qPCR instrument and analysis using Protein Thermal Shift™software (Life Technologies Corp.). The run time conditions were: atemperature hold at 20° C. for one minute followed by a thermal ramp of0.05° C. with continuous data collection to 95° C., followed by a 1minute hold at the final temperature.

By inspection of FIG. 3A, melt curve data for the triplicate samplesusing the BODIPY® 499/508 maleimide is shown in FIG. 3A-I, while thetriplicate set using Sypro® Orange is shown in FIG. 3A-II shows no dyeresponse as a function of temperature. It is clear from the data thatSypro® Orange does not work effectively with this protein to producemelt curve results. From the set of data in FIG. 3A-I, or the firstderivative data shown in FIG. 3B-1, a T_(m) of 56° C. may be determinedfor β-lactoglobulin. FIG. 4A is derived as blown-up section of FIG. 3A,while FIG. 4B is the same derivative data as shown in FIG. 3A. It isclear from inspection of FIG. 4A that that Sypro® Orange additionallyproduces a high-background. As previously mentioned, one of theattributes of dipyrrometheneboron difluoride dye of formula (1) are thatthey produce a negligible intrinsic background fluorescent signal.Additionally, the enhanced stability against photobleaching for dyes offormula (1) produces a clear, S-shaped curve.

FIGS. 5A and 5B depicts protein melt data and first derivative of themelt data, respectively, for a sample of bovine α-chymotrypsin (Sigma PNC4129). Triplicate dye-protein samples for α-chymotrypsin-BODIPY®499/508 maleimide and α-chymotrypsin-Sypro® Orange were prepared and runas described for β-lactoglobulin. As can be seen in FIG. 5A-I and FIG.5B-II, BODIPY® 499/508 maleimide produces melt curve data, and firstderivative of the melt curve data, respectively from which a T_(m) of59° C. may be determined for α-chymotrypsin. Similarly to the dataproduced for β-lactoglobulin, there are no melt curve results producedfor α-chymotrypsin using Sypro® Orange. As can be seen in FIG. 6A, whichis derived as blown-up section of FIG. 5A, there is no appreciablebackground for Sypro® Orange for this analysis.

In FIGS. 7A and 7B an additional example of the use of BODIPY® 499/508maleimide with a cysteine-containing protein is shown for exonuclease I.(New England Biolabs M0293L; 20 units/ul). Triplicate dye-proteinsamples for exonuclease I-BODIPY® 499/508 maleimide and exonucleaseI-Sypro® Orange were prepared and run as described for β-lactoglobulin.As can be seen in FIG. 7A-I and FIG. 7B-II, BODIPY® 499/508 maleimideproduces melt curve data, and first derivative of the melt curve data,respectively from which a T_(m) of 40-45° C. may be determined forexonuclease I. Similarly to the data produced for β-lactoglobulin andα-chymotrypsin, there are no melt curve results produced for exonucleaseI using Sypro® Orange. As can be seen in FIG. 8A, which is derived asblown-up section of FIG. 7A, there is no appreciable background forSypro® Orange for this analysis.

Regarding FIGS. 9A and 9B, which are melt curve data and firstderivative melt curve data, respectively, a sample set was run forlysozyme (Sigma L3790; 10 mg/ml), which is a protein known to have nocysteine residues. Triplicate dye-protein samples for lysozyme-BODIPY®499/508 maleimide and lysozyme-Sypro® Orange were prepared and run asdescribed for β-lactoglobulin. In FIG. 9A-1 and FIG. 9B-1, there is nomelt curve peak for the sample assayed using BODIPY® 499/508 maleimide,but an apparent peak and derivative peak in FIG. 9A-II and FIG. 9B-IIfor the samples run using Sypro® Orange. For the melt curve datagenerated using Sypro® Orange, a T_(m) of 73° C. could be determined forlysozyme.

Finally, FIG. 10A and FIG. 10B depict the melt curve data for aCAAX-containing membrane protein. As one of ordinary skill in the art isapprised, CAAX refers to a C-terminal prenylation signal sequence, whichcontains a cysteine residue. The membrane protein is suspended in asolution containing a surfactant mixture; 40 mM MES pH 6.5, 400 mM NaCl,and 0.1% DDM, in order to stabilize the membrane protein in vitro. Theconditions for the assay were as previously described forβ-lactoglobulin, with a total of 5 μg of the membrane protein used perassay. FIG. 10A-I and FIG. 10B-I are the protein melt data, and firstderivative data, respectively for the assay run using BODIPY® 499/508maleimide. The data indicate a T_(m) of about 45° C. In FIG. 10A-II andFIG. 10B-II, which are the protein melt data, and first derivative data,respectively for the assay run using Sypro® Orange, there is a highsloping baseline, as well as problems with reproducibility of thereplicates. The data suggest that even in the presence of highconcentrations of a surfactant, that dipyrrometheneboron difluoride dyeof formula (1) may give consistent results for protein melt analysis.

While the present teachings have been described in conjunction withvarious embodiments and examples, it is not intended that the presentteachings be limited to such embodiments or examples. On the contrary,the present teachings encompass various alternatives, modifications, andequivalents, as will be appreciated by those of skill in the art. Theteachings should not be read as limited to the described order orelements unless stated to that effect. It should be understood thatvarious changes in form and detail may be made without departing fromthe scope of the present teachings, including the order and arrangementof disclosed method steps. Therefore, all embodiments that come withinthe scope and spirit of the present teachings and equivalents theretoare claimed.

What is claimed is:
 1. A method comprising: (a) forming a samplesolution mixture comprising at least one protein and a dye of formula(I):

wherein: R₁ to R₆ is independently selected from the group consistingof: hydrogen, halogen, —(CH₂)_(n)CO₂H (wherein n=0 to 6), —(CH₂)_(n)CO₂R(wherein n=0 to 6), cycloakyl, alkyl (1-5 carbons), aryl, heteroaryl,arylalkyl (wherein the alkyl portion is 1-5 carbon atoms), alkenyl,azido, alkynyl, and sulfo, alone or in combination, wherein o, m and pare independently selected from the group consisting of: hydrogen,halogen, —(CH₂)_(n)CO₂H (wherein n=0 to 6), —(CH₂)_(n)CO₂R (wherein n=0to 6), cycloakyl, alkyl (1-5 carbons), aryl, heteroaryl, arylalkyl(wherein the alkyl portion is 1-5 carbon atoms), alkenyl, azido,alkynyl, sulfo, and a maleimidyl substituent having the structure:

alone or in combination, with the proviso that the maleimidylsubstituent occurs in formula (I) once and only once, wherein an alkenylsubstituent is substituted or unsubstituted, wherein the alkenyl groupis ethenyl, dienyl, or trienyl, wherein the one or more substituents foran alkenyl group is selected from the group consisting of: hydrogen,halogen, alkyl (1-5 carbon atoms), cyano, carboxylate ester,carboxamide, aryl, heteroaryl, and any combination thereof, wherein anaryl is selected from phenyl, 1-naphthyl, 2-naphthyl, 1-pyrenyl,9-anthryl, pyridyl, quinolyl, and alkoxy derivatives thereof, wherein anaryl or heteroaryl group is substituted or unsubstituted, wherein theone or more substituents for an aryl or heteroaryl group is selectedfrom the group consisting of: hydrogen, halogen, —(CH₂)_(n)CO₂H (whereinn=0 to 6), —(CH₂)_(n)CO₂R (wherein n=0 to 6) alkyl (1-5 carbons),alkoxy(wherein the alkyl portion is 1˜4 carbon atoms), and anycombination thereof, wherein any alkyl or arylalkyl group is substitutedor unsubstituted, wherein the one or more substituents for an alkyl orarylalkyl group is selected from the group consisting of: hydrogen,halogen, —(CH₂)_(n)CO₂H (wherein n=0 to 6), —(CH₂)_(n)CO₂R (wherein n=0to 6), cycloakyl, alkyl (1-5 carbons), aryl, heteroaryl, arylalkyl(wherein the alkyl portion is 1-5 carbon atoms), alkenyl, azido,alkynyl, and sulfo, alone or in combination, wherein the alkyl group inany substituent of an aryl group may be further substituted by an esteror amide substituent, and wherein L is an optionally present linker andwhen present L is selected from alkyl (1-6 carbons), and heteroalkyl,(1-6 atoms); and (b) applying a controlled heating to the samplesolution mixture; and (c) measuring fluorescence emitted over atemperature range.
 2. The method of claim 1, wherein the dye in theprotein-dye mixture is4,4-difluoro-1,3,5,7-tetramethyl-8-(4-maleimidylphenyl)-4-bora-3a,4a-diaza-s-indacene.3. The method of claim 1, wherein the dye in the protein-dye mixture is4,4-difluoro-3,5-bis(4-methoxyphenyl)-8-(4-maleimidylphenyl)-4-bora-3a,4a-diaza-s-indacene.4. The method of claim 1, wherein the controlled heating is a thermalramp.
 5. The method of claim 4, wherein the thermal ramp is betweenabout 20° C. to about 95° C.
 6. The method of claim 1, wherein thecontrolled heating is isothermal heating.
 7. The method of claim 1,wherein the sample solution mixture further comprises a buffer.
 8. Themethod of claim 1, wherein the sample solution mixture further comprisesa surfactant.
 9. The method of claim 8, wherein the surfactant is at orabove the critical micelle concentration.
 10. The method of claim 1,wherein the sample solution mixture further comprises a polyol.
 11. Themethod of claim 10, wherein the polyol is glycerol.
 12. The method ofclaim 10, wherein the polyol is a polysaccharide.